West Nile virus (WNV) is a mosquito-borne flavivirus that infects humans, horses, and birds. The virus is transmitted to humans and several animal species through mosquitoes that acquire the virus by feeding on infected birds. The virus is indigenous to Africa, Asia, Europe, and Australia, and has recently caused large epidemics in the Western Hemisphere, including in Europe and the United States. WNV was first detected in North America in 1999 during an epidemic of meningoencephalitis in New York City. WNV seroprevalence studies in Queens, New York showed evidence of prior infection in 2.6% of the population, age 5 or older. During 1999-2002, the virus extended its range throughout much of the eastern United States. The range of WNV infections within the Western Hemisphere is expected to continue to expand.
Human WNV infections are often subclinical but clinical infections can range in severity from uncomplicated fever to fatal meningoencephalitis. The incidence of severe neuroinvasive disease and death increases with age. Epidemics of WNV encephalitis and meningitis raise concerns that transmission of WNV may occur through voluntary blood donations.
As with other flaviviruses, WNV possesses a single-stranded plus-sense RNA genome of approximately 10,000 nucleotides. The genome contains a single open reading frame (ORF) of about 10,300 nucleotides that encodes a polyprotein that is proteolytically processed into 10 mature viral proteins, in the order of NH2—C—PrM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-COOH. The three structural proteins, capsid (C), membrane (PrM), and envelope (E), are encoded within the 5′ portion of the ORF, while the seven nonstructural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5, are encoded within the 3′ portion. The boundaries of these proteins, numbered relative to the nucleotide sequence of WNV, strain NY99, are as follows: C, 97-465; pr, 466-741; M, 742-986; E, 987-2469; NS1, 2470-3525; NS2A, 3526-4218; NS2B, 4219-4611; NS3, 4612-6458; NS4A, 6459-6915; NS4B, 6916-7680; NS5, 7681-10395. For a review of WNV and its molecular biology and structure, see, Brinton, M. A., Ann. Rev. Microbiol. (2002) 56:371-402; and Lanciotti et al., Science (1999) 286:2333-2337.
To date, no effective prevention or treatment of WNV infection exists. Currently, public education and mosquito abatement programs are used to curb transmission of the virus. However, rapid intervention is critical in order to reduce the risk to humans. Traditionally, detection of virus has been accomplished by testing mosquitoes and dead birds for the presence of virus using cell culture methods and immunoassay techniques. However, these methods are extremely time consuming and can take a week or more to complete.
The diagnosis of WNV infection in humans can be established by the presence of WNV IgM antibody in serum or cerebrospinal fluid (CSF), increases in WNV antibody detected by ELISA or WNV neutralizing antibody. However, confirmation of the type of infecting virus is possible only by detection of a fourfold or greater rise in virus-specific neutralizing antibody titers in either CSF or serum by performing plaque reduction neutralization assays with several flaviviruses. Virus isolation in cell culture from CSF and serum has generally been unsuccessful, likely due to the low level and short-lived viremia associated with infection. Additionally, most immunological tests are indirect, and nonspecific antigen-antibody reactions can occur and result in false-positive determinations. Hence, immunological methods for successfully diagnosing WNV infection are greatly needed.
Attempts have been made to develop vaccines for WNV. In particular, killed virus vaccines, a live attenuated chimeric virus vaccine and passive immunization with WNV-immune serum have been studied. Tesh et al., Emerg. Infect. Dis. (2002) 8:1392-1397; Malkinson et al., Ann. N.Y. Acad. Sci. (2001) 951:255-261; Monath et al., Curr. Drug Targets Infect. Disord. (2001) 1:37-50. The WNV E protein has been produced recombinantly and administered to mice. See, U.S. Patent Publication No. 2003/0148261; Wang et al., J. Immunol. (2001) 167:5273-5277. Wang et al., Ann. NY Acad. Sci. (2001) 951:325-327 report the passive immunization of mice with rabbit anti-E protein sera. PCT Publication No. WO 02/083903 describes the use of WNV peptides in vaccines.
DNA vaccines including either WNV PrM-E or C have also been studied. See, Davis et al., J. Virol. (2001) 75:4040-4047; Chang et al., Ann. NY Acad. Sci. (2001) 951:272-285; Yang et al., J. Infect. Dis. (2001) 184:809-816; U.S. Patent Publication Nos. 2003/0022849, 2003/0104008 and 2003/0091595. For example, Davis et al. J. Virol. (2001) 75:4040-4047 describes a DNA construct encoding PrM and E proteins under the control of the Japanese encephalitis virus signal sequence. The recombinant antigen expressed by the construct is assembled and secreted in the form of extracellular subviral particles. U.S. Patent Publication No. 2002/0164349 reports the recombinant production of a WNV capsid protein and immunization using a plasmid encoding the capsid.
Nevertheless, there remains an urgent need for immunogenic reagents for use in vaccines and as diagnostics for WNV.